Funded Projects

Start of funding 01.01.2001

Structural analysis of alpha-synuclein fibrillogenesis

Prof. Dr. rer. nat. Christian Haass
Ludwig-Maximilians-University of Munich
Medical Faculty Adolf-Butenandt-Institute, Department of Metabolic Biochemistry

Prof. Dr. Klaus Beyer
Ludwig-Maximilians-University of Munich
Medical Faculty Adolf-Butenandt-Institute, Department of Metabolic Biochemistry

Prof. Dr. Charles Glabe
University of California, Irvine
Department of Molecular Biology and Biochemistry

The formation of insoluble a-synuclein (alphaSYN) fibrils contributes to Parkinson's disease. The central portion of alphaSYN (called NAC) is critically involved in this process. We have developed an in vitro alphaSYN fibrillization assay allowing monitoring of this process using circular dichroism and nuclear magnetic resonance spectroscopy. We attempt to identify key amino acid side chains essential for alphaSYN fibrillization by combined biophysical studies and site-directed mutagenesis. Peptides that retain the structural properties of the biologically relevant full-length protein will be synthesized and characterized. The solution structure of NAC during fibrillogenesis will be monitored. Structural information on alphaSYN aggregation may produce a template for rational design of anti-aggregation drugs for Parkinson's disease.

Final report:
Fibrillar deposits of α-synuclein represent the hallmark feature of Parkinson’s disease and of related synucleinopathies. The present project aimed at an advanced understanding of self aggregation and membrane binding of α-synuclein. We were able to show that the protein not only binds to vesicles with negative surface charges but also to electroneutral vesicles with interfacial packing defects. These conclusions were drawn from the binding thermodynamics and from the concomitant folding of the protein as shown by calorimetry and by optical techniques (1). Our results suggest that the protein is involved in stabilizing the synaptic reserve pool. A number of peptides were synthesized for an investigation of the folding and aggregation propensities of isolated protein domains, on the basis of a current NMR derived structure of helical α-synuclein. It turned out that only a sequence comprising residues 41 – 55 display a tendency to form a stable helix spontaneously in the presence of small phospholipid vesicles. Micelles of the anionic detergent SDS, however, were able to induce unusually stable helical structures in the peptide fragments 1 – 25 and 51 – 65. This observation casts the results of the NMR studies into doubt where the SDS micelle has been usually employed in order to meet the conditions for high resolution work (2-5). For the future it is planned to study the interaction of selected peptides with the full length protein. A detailed investigation of peptide – protein interactions appears promising regarding recent reports on transient α-synuclein structures with the capability of inhibiting self aggregation of the protein (6,7).

References:

1. Nuscher, B., Kamp, F., Mehnert, T., Odoy, S., Haass, C., Kahle, P. J., and Beyer, K. (2004) J Biol Chem 279, 21966-21975.
2. Chandra, S., Chen, X., Rizo, J., Jahn, R., and Südhof, T. C. (2003) J. Biol. Chem. 278, 15313-15318.
3. Eliezer, D., Kutluay, E., Bussell, R., Jr., and Browne, G. (2001) J Mol Biol 307, 1061-1073.
4. Bussell, R., Jr., and Eliezer, D. (2003) J. Mol. Biol. 329, 763-778.
5. Ulmer, T. S., Bax, A., Cole, N. B., and Nussbaum, R. L. (2005) J Biol Chem 280, 9595-9603.
6. Bertoncini, C. W., Jung, Y. S., Fernandez, C. O., Hoyer, W., Griesinger, C., Jovin, T. M., and Zweckstetter, M. (2005) Proc Natl Acad Sci U S A 102, 1430-1435.
7. Li, W., West, N., Colla, E., Pletnikova, O., Troncoso, J. C., Marsh, L., Dawson, T. M., Jakala, P., Hartmann, T., Price, D. L., and Lee, M. K. (2005) Proc Natl Acad Sci U S A 102, 2162-2167.